Technical validation of an RT-qPCR in vitro diagnostic test system for the determination of breast cancer molecular subtypes by quantification of ERBB2, ESR1, PGR and MKI67 mRNA levels from formalin-fixed paraffin-embedded breast tumor specimens

• Published in: BMC cancer 2016-07, Vol.16 (1), p.398-398, Article 398
• Main Authors: Laible, Mark, Schlombs, Kornelia, Kaiser, Katharina, Veltrup, Elke, Herlein, Stefanie, Lakis, Sotiris, Stöhr, Robert, Eidt, Sebastian, Hartmann, Arndt, Wirtz, Ralph M, Sahin, Ugur
• Format: Article
• Language: English
• Subjects: Biomarkers; Breast cancer; Breast Neoplasms - classification; Breast Neoplasms - diagnosis; Breast Neoplasms - genetics; Cancer therapies; Classification; Diagnosis; Diagnostic tests; Diagnostic Tests, Routine - methods; Estrogen Receptor alpha - genetics; Feasibility Studies; Female; Genes; Humans; Ki-67 Antigen - genetics; Laboratories; Medical prognosis; Messenger RNA; Metastasis; Paraffin Embedding; Pathology; Patients; Polymerase chain reaction; Properties; Real-Time Polymerase Chain Reaction - methods; Receptor, ErbB-2 - genetics; Receptors, Progesterone - genetics; Reproducibility of Results; RNA, Messenger - metabolism; Sensitivity and Specificity; Technical Advance; Tissue Fixation; Tumors
• ISSN: 1471-2407; 1471-2407
• DOI: 10.1186/s12885-016-2476-x

MammaTyper is a novel CE-marked in vitro diagnostic RT-qPCR assay which assigns routinely processed breast cancer specimens into the molecular subtypes Luminal A-like, Luminal B-like (HER2 positive or negative), HER2 positive (non-luminal) and Triple negative (ductal) according to the mRNA expression of ERBB2, ESR1, PGR and MKI67 and the St Gallen consensus surrogate clinical definition. Until now and regarding formalin-fixed, paraffin-embedded material (FFPE), this has been a task mostly accomplished by immunohistochemistry (IHC). However the discrepancy rates of IHC for the four breast cancer biomarkers are frequently under debate, especially for Ki-67 which carries the highest degree of inter- and even intra-observer variability. Herein we describe a series of studies in FFPE specimens which aim to fully validate the analytical performance of the MammaTyper assay, including the site to site reproducibility of the individual marker measurements. Tumor RNA was extracted with the novel RNXtract RNA extraction kit. Synthetic RNA was used to assess the sensitivity of the RNXtract kit. DNA and RNA specific qPCR assays were used so as to determine analyte specificity of RNXtract. For the assessment of limit of blank, limit of detection, analytical measurement range and PCR efficiency of the MammaTyper kit serial dilutions of samples were used. Analytical precision studies of MammaTyper were built around two different real time PCR platforms and involved breast tumor samples belonging to different subtypes analyzed across multiple sites and under various stipulated conditions. The MammaTyper assay robustness was tested against RNA input variations, alternative extraction methods and tumor cell content. Individual assays were linear up to at least 32.33 and 33.56 Cqs (quantification cycles) for the two qPCR platforms tested. PCR efficiency ranged from 99 to 109 %. In qPCR platform 1, estimates for assay specific inter-site standard deviations (SD) were between 0.14 and 0.20 Cqs accompanied by >94 % concordant single marker assignments for all four markers. In platform 2, the inter-site SD estimates were between 0.40 and 0.66 Cqs while the concordance for single marker assignments was >94 % for all four markers. The agreement reached between the two qPCR systems located in one site was 100 % for ERBB2, 96.9 % for ESR1, 97.2 % for PGR and 98.6 % for MKI67. RT-qPCR for individual markers was stable up to a 64-fold dilution for a typical clinical sample. There was n